Gene silencing through RNA interference (RNAi) has revolutionized the study of gene 98 function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) 99 RNAi has many times proven to be difficult to achieve. Most of the negative results have been 100 anecdotal and the positive experiments have not been collected in such a way that they are 101 possible to analyze. In this review, we have collected detailed data from more than 150 102 experiments including all to date published and many unpublished experiments. Despite a 103 large variation in the data, trends that are found are that RNAi is particularly successful in the 104 family Saturniidae and in genes involved in immunity. On the contrary, gene expression in 105 epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding 106 dsRNA requires high concentrations for success. Possible causes for the variability of success 107 in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further 108 investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the 109 innate immune response. Our general understanding of RNAi in Lepidoptera will be further 110 aided in the future as our public database at http://insectacentral.org/RNAi will continue to 111 gather information on RNAi experiments.

Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for hostinduced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE protein HvSNAP34 in three types of durable, race-nonspecific resistance was observed.

Obligate biotrophic pathogens of plants must circumvent or counteract defenses to guarantee accommodation inside the host. To do so, they secrete a variety of effectors that regulate host immunity and facilitate the establishment of pathogen feeding structures called haustoria. The barley powdery mildew fungus Blumeria graminis f. sp. hordei produces a large number of proteins predicted to be secreted from haustoria. Fifty of these Blumeria effector candidates (BEC) were screened by host-induced gene silencing (HIGS), and eight were identified that contribute to infection. One shows similarity to β-1,3 glucosyltransferases, one to metallo-proteases, and two to microbial secreted ribonucleases; the remainder have no similarity to proteins of known function. Transcript abundance of all eight BEC increases dramatically in the early stages of infection and establishment of haustoria, consistent with a role in that process. Complementation analysis using silencing-insensitive synthetic cDNAs demonstrated that the ribonuclease-like BEC 1011 and 1054 are bona fide effectors that function within the plant cell. BEC1011 specifically interferes with pathogen-induced host cell death. Both are part of a gene superfamily unique to the powdery mildew fungi. Structural modeling was consistent, with BEC1054 adopting a ribonuclease-like fold, a scaffold not previously associated with effector function.

HighlightWheat plants transiently or stably accumulating antisense or double-stranded RNA, which are directed against essential genes of the Fusarium head blight fungus F. culmorum, were more resistant against the disease.

To study protein ubiquitination pathways in the interaction of barley (Hordeum vulgare) with the powdery mildew fungus (Blumeria graminis), we measured protein turnover and performed transient-induced gene silencing (TIGS) of ubiquitin and 26S proteasome subunit encoding genes in epidermal cells. Attack by B. graminis hyperdestabilized a novel unstable green fluorescent protein fusion that contains a destabilization domain of a putative barley 1-aminocyclopropane-1-carboxylate synthase, suggesting enhanced protein turnover. Partial depletion of cellular ubiquitin levels by TIGS induced extreme susceptibility of transformed cells toward the appropriate host pathogen B. graminis f. sp hordei, whereas papilla-based resistance to the nonhost pathogen B. graminis f. sp tritici and host resistance mediated by the mlo gene (for mildew resistance locus O) remained unaffected. Cells were rescued from TIGS-induced ubiquitin depletion by synthetic genes encoding wildtype or mutant barley monoubiquitin proteins. The strongest rescue was from a gene encoding a K63R mutant form of ubiquitin blocked in several ubiquitination pathways while still allowing Lys-48-dependent polyubiquitination required for proteasomal protein degradation. Systematic RNA interference of 40 genes encoding all 17 subunits of the proteasome 19S regulatory particle failed to induce hypersusceptibility against B. graminis f. sp hordei. This suggests a role for Lys-48-linked protein polyubiquitination, which is independent from the proteasome pathway, in basal host defense of barley.

No abstract

Background: Non-host resistance, NHR, to non-adapted pathogens and quantitative host resistance, QR, confer durable protection to plants and are important for securing yield in a longer perspective. However, a more targeted exploitation of the trait usually possessing a complex mode of inheritance by many quantitative trait loci, QTLs, will require a better understanding of the most important genes and alleles.